This thesis discusses the development of time resolved fluorescence imaging techniques and their use in the study of cellular signalling pathways, in particular the ability to perform multiplexed imaging of a number of pathways in live cells. These techniques are applied to investigate chemotaxis, the ability of cells to migrate directionally in response to a chemoattractant gradient, which requires precise spatiotemporal coordination of signalling events. Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) biosensors to map protein-protein interactions in live cells. The development of a software tool for the global analysis of large FLIM datasets is presented which allows simultaneous analysis of hundreds of FLIM images in minutes and the use of complex models, for example a four-exponential model of an ECFP FRET system, with relatively low photon-count data. Live cell imaging with optimised FRET biosensors is used to investigate the role of Phospholipase C epsilon (PLCε) in fibroblast chemotaxis. It is demonstrated that PLCε-null fibroblasts show a compromised chemotactic response to platelet derived growth factor and spatial defects in Rac1 activation and phosphoinositide signalling. The ability to image multiple functional reporters simultaneously in a single cell is desirable when investigating complex signalling networks with significant cross-talk such as chemotaxis. A number of approaches for multiplexed measurements are investigated, in particular using homo-FRET between two spectrally identical fluorophores, which presents a promising approach to reduce the spectral bandwidth compared to conventional hetero-FRET biosensors. The optimisation and automation of a to perform multiplexed time-resolved fluorescence anisotropy imaging of homo-FRET biosensors is discussed. The development and multiplexed imaging of homo-FRET reporters for phosphoinositide signalling using a polarisation resolved confocal time correlated single photon counting (TCSPC) microscope is presented. Potential approaches for multiplexed imaging three functional reporters are discussed.